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First published online 28 October 2008
doi: 10.1242/jcs.032441

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The Rip11/Rab11-FIP5 and kinesin II complex regulates endocytic protein recycling

¹ Department of Cellular and Developmental Biology, School of Medicine, University of Colorado Health Sciences Center, 12801 E. 17th Avenue, Aurora, CO 80045, USA
² Department of Biological Sciences, Imperial College London, London, SW7 2AZ, UK
³ Departments of Surgery and Cell and Developmental Biology, Vanderbilt University and the Nashville VA Medical Center, Nashville, TN 37232, USA

View larger version (65K): [in this window] [in a new window]   Fig. 1. Rip11/FIP5 is enriched in peripheral recycling endosomes. (A-F) HeLa cells were plated on collagen-coated glass coverslips fixed and stained with anti-TfR (B,C,E,F, red) or anti-Rip11/FIP5 (A,C,D,F, green) antibodies. Yellow in C and F represents overlap between TfR and Rip11/FIP5. (D-F) High-magnification images of the same cell. Arrowheads in D-F indicate organelles where TfR and Rip11 colocalize. Arrows in D-F indicate organelles where Rip11/FIP5 is enriched in subdomains. Scale bars: 5 µm (A) and 1 µm (D). (G,H) HeLa cells were incubated with transferrin-HRP for 45 minutes at 37°C before being processed for immunoelectron microscopy. Anti-Rip11/FIP5 antibodies were detected using 10 nm gold. The electron-dense DAB reaction product indicates the presence of Tf. Clusters of Tf-positive endosomes that are also positive for Rip11/FIP5 are visible. Scale bars: 500 nm. Inset in H is a higher magnification image of endocytic tubules. Inset in G shows a vesicular part of an early endosome that is negative for anti-Rip11/FIP5 antibodies. Scale bar: 100 nm. Arrows indicate Tf-HRP-containing endosomes that are also positive for Rip11-gold. Arrowheads indicate Rip11 clusters on Tf-HRP endosomes.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Rip11/FIP5 knockdown increases Tf uptake. (A) HeLa cells treated with mock siRNA, Rip11/FIP5 siRNA1 or Rip11/FIP5 siRNA2 were incubated at 37°C with Tf-Alexa488. Cells were then washed, fixed and internalized Tf-Alexa488 measured by flow cytometry. The data shown are the mean ± s.d. of three independent experiments. Asterisks indicate time points that are significantly different from mock-treated control at P<0.025. (B) Mock-treated or Rip11/FIP5 siRNA-treated HeLa cells were fixed and incubated with 80 µg/ml Tf-Alexa488 in the absence or presence of 0.4% saponin. Cells were then washed and plasma-membrane-associated (non-permeabilized cells, see figure) and total cellular (permeabilized cells, not shown) Tf-Alexa488 measured by flow cytometry. The data shown are the mean ± s.d. of three independent experiments. (C) Mock- and Rip11/FIP5 siRNA1-treated HeLa cells were incubated at 37°C for 30 minutes with Tf-Alexa488. Cells were then washed and incubated at 37°C with unlabeled Tf. Cell-associated Tf-Alexa488 was measured by flow cytometry and is shown as arbitrary units. The data shown are the mean ± s.d. of three independent experiments. *P<0.01. (D) The rates of Tf uptake and recycling calculated from the data presented in A and C. The data shown are the half-life of uptake and recycling (T1/2) as well as the amount of Tf-Alexa488 internalized and recycled (arbitrary fluorescence units per minute). Data are the mean ± s.d. calculated from three different experiments. *P<0.02.

View larger version (93K): [in this window] [in a new window]   Fig. 3. Rip11/FIP5 knockdown increases colocalization of Tf with TfR. Mock-treated (A-F) or Rip11 siRNA1-treated (J-L) HeLa cells were loaded for 30 minutes at 37°C with 5 µg/ml Tf-Alexa594 (B,C,E,F,H,I,K,L, red). Cells were then washed fixed and stained with anti-TfR antibodies (A,C,D,F,G,I,J,L, green). Yellow in C,F,I and L represents the overlap between Tf-Alexa594 and TfR. D-F and J-L are higher magnification images of boxed areas of mock-treated and Kif3B siRNA1-treated cells, respectively. Scale bars: 5 µm (A,G) and 1 µm (D,J). Arrowheads in D-F indicate organelles enriched in Tf-Alexa594. Arrows in D-F indicate organelles containing TfR but little Tf-Alexa594. Arrowheads in J-L point to large organelles enriched in TfR and Tf-Alexa594. Numbers in A and G give the percentage overlap between TfR and Tf-Alexa594. The data are mean ± s.d. overlap in 25 randomly chosen cells from three different experiments.

View larger version (77K): [in this window] [in a new window]   Fig. 4. Rip11/FIP5 knockdown increases colocalization of Tf with early endosomes. Mock-treated (A-C) or Rip11 siRNA1-treated (D-F) HeLa cells were incubated for 30 minutes with Tf-Alexa488 (A,D and green in C,F). Cells were then washed, fixed and stained with anti-EEA1 antibody (B,E and red in C,F). Yellow colour represents the extent of Tf and EEA1 overlap. Asterisks indicate early endosomes. Insets in C and F are higher magnification images. Numbers in C and F represent the extent of colocalization of Tf-Alexa488 with EEA1. The numbers are the mean ± s.d. from ten random cells. Scale bars: 4 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Kinesin II mediates Rip11/FIP5 binding to microtubules. (A) Glutathione beads coated with GST alone, GST-Kif3A-tail, GST-Kif3B-tail or GST-Kif3C-tail were incubated with HeLa cell Triton X-100 lysates. Beads were then washed and bound Rip11/FIP5 or RCP/FIP1 was analyzed by immunoblotting. The lower molecular size band present only in the GST lane represents a non-specific band since it did not disappear when we use lysates treated with Rip11/FIP5 siRNA (data not shown). (B) Glutathione beads coated with GST alone, GST-Kif3A-tail, GST-Kif3B-tail or GST-Kif3C-tail were incubated with recombinant purified 6His-Rip11/FIP5. Bound 6His-Rip11/FIP5 was detected by immunoblotting. (C) Glutathione beads coated with either GST alone or GST-Kif3B-tail were incubated with Rip11/FIP5(490-652) in the presence or absence of recombinant Rab11a. Bound Rip11/FIP5(490-652) was detected by immunoblotting. (D,E) Rip11/FIP5 binding to microtubules analyzed using microtubule sedimentation assay (see Materials and Methods). Where indicated, assays were performed in the presence off either 5 mM ATP or recombinant Kif3B-tail protein. In the bottom panel, mock-treated or Kif3B siRNA-treated lysates were used in the assay.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Kif3B knockdown increases Tf uptake. (A) Mock-treated or Kif3B siRNA1-treated HeLa cells incubated at 37°C with Tf-Alexa488. Internalized Tf-Alexa488 was measured by flow cytometry. The data shown are the means of two independent experiments. (B) Mock-treated or Kif3B siRNA-treated HeLa cells were fixed with 4% paraformaldehyde and incubated with 80 µg/ml of Tf-Alexa488 in the absence of saponin. Plasma-membrane-associated Tf-Alexa488 was measured by flow cytometry. The data shown are the means ± s.d. of three independent experiments. (C) Mock-treated or Kif3B siRNA1-treated HeLa cells incubated at 37°C for 30 minutes with Tf-Alexa488. Cells were incubated with unlabeled Tf. Cell-associated Tf-Alexa488 was then measured by flow cytometry and is shown as arbitrary fluorescence units. The data shown are the mean ± s.d. of three independent experiments. *P<0.025.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Kif3B knockdown results in peripheral accumulation of TfR. (A-C) Kif3B siRNA1-treated cells incubated for 30 minutes with Tf-Alexa488 (A,C, green). Cells were then fixed and stained with anti-EEA1 antibodies (B,C, red). Asterisks in inset in C indicate the early endosomes containing Tf. Yellow in C represents the overlap between Tf-Alexa488 and EEA1. Scale bar: 5 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 8. Rip11/FIP5 and Kif3B regulate the same recycling pathway. (A-C) Mock-treated cells (A) or cells treated with Kif3B siRNA1 (B) or Kif3B and Rip11/FIP5 siRNA1 (C) were incubated at 37°C for 30 minutes with Tf-Alexa488. Scale bars: 5 µm. (D) Proposed model of roles of Rip11/FIP5 and kinesin II in mediating transport to the slow storage pool of endocytic recycling.

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